The use of organisms that have advanced genetic systems as hosts for nucleic acid molecules isolated from a variety of species allows for the manipulation of the isolated nucleic acid sequences in the host. The ability to engineer organisms by cloning and modifying chromosomes and genomes in exogenous hosts is limited, however, by the size limitation on nucleic acid molecules that can be transferred to species such as yeast that have tractable genetics.
Nucleic acids cloned by conventional methods generally contain no more than a few genes, although larger nucleic acids (e.g., DNA) have been transferred into host cells. For example, the 16 kb mouse mitochondrial genome has been cloned in E. coli (Itaya et al., Nat Methods 5, 41 (2008); Yoon and Koob, Nucleic Acids Res 31, 1407 (2003)), Bacillus subtilis (Itaya et al., Nat Methods 5, 41 (2008); Yoon and Koob, Nucleic Acids Res 31, 1407 (2003)), and yeast (Wheeler et al., Gene 198, 203 (1997)). The 139 kb maize chloroplast genome has been cloned in yeast (Gupta and Hoo, Plant Mol Biol 17, 361 (1991), and the 135 kb rice chloroplast genome has been cloned in B. subtilis (Itaya et al., Nat Methods 5, 41 (2008)). About 10% of the 1.8 Mb Haemophilus influenzae genome has been cloned as episomal elements in E. coli (Smailus et al., Syst Synth Biol; 1, 139 (2007)). The 3.5 Mb Synechocystis PCC6803 genome was inserted in three noncontiguous regions into the B. subtilis genome, with the exception of the two ribosomal RNA operons (Itaya et al., PNAS USA 102, 15971 (2005)). A complete synthetic 0.6 Mb Mycoplasma genitalium genome has been assembled in yeast as a circular yeast centromeric plasmid (YCp) (Gibson et al., Science 319, 1215 (2008); Gibson et al., PNAS USA, 105(51):20404-9 (2008)).
U.S. Pat. No. 6,670,154 describes methods for converting modified bacterial genomes into artificial yeast chromosomes by fusing the bacteria with yeast that linearize the modified genomes. U.S. Patent Application Publication No. 2005/0019924 describes nucleic acids and methods for introducing prokaryotic genomes into eukaryotic cells as circular molecules and conversion into artificial chromosomes. WO 02/057437 describes YAC vectors containing cytomegalovirus (CMV) genomes. U.S. Pat. No. 7,083,971 describes a recombinatorial approach and system for cloning, manipulating, and delivering large nucleic acid segments. U.S. Patent Application Publication No. 2005/0003511 and Bradshaw et al., Nucleic Acids Research, 23, 4850-56 (1995) describe yeast-bacterial shuttle vectors for cloning large regions of DNA by homologous recombination.
The disclosed cloning and manipulation methods, however, are limited by the size of donor nucleic acids that can be transferred into a host cell, and do not provide for manipulating and/or transferring a nucleic acid molecule propagated in a host cell back into a recipient cell that is related to the donor, nor do they address incompatibility issues among different cell types used in cloning with regard to foreign nucleic acids. Additional methods are needed for cloning large nucleic acids, such as chromosomes or genomes, into alternate heterologous hosts, for manipulating the sequences of large nucleic acids in alternate hosts, and for transferring manipulated genomes back into recipient organisms that are similar to the donor organism, for example, organisms of the same genus (for example, from prokaryotic to eukaryotic cells and back).
To date, the barriers to transferring large nucleic acids, such as chromosomes and genomes, between organisms of different species or different genuses have not been overcome. For example transfer of the nucleic acids between species can be toxic to host, donor, and/or recipient cells. Manipulation and propagation of nucleic acids in organisms of different species, genuses, or groups and from prokaryotic to eukaryotic cells and back can also cause instability of the nucleic acids and inhibit their activation, such as expression of genes from the nucleic acids.